This invention is in the general field of biochemical engineering. More specifically, the subject invention relates to the preparation of biologically active recombinant lipophilic proteins such as human interferons and interleukin-2. Still more specifically, the subject invention relates to an improved process for the production and recovery of lipophilic proteins from genetically transformed host organisms, lipophilic protein preparations of relatively high purity, and therapeutically acceptable formulations thereof.
Interleukin-2 (IL-2) is a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli. IL-2 was first described by Morgan, D. A., et al., Science (1976) 193:1007-1008 and was then designated T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. Now renamed as interleukin-2, IL-2 later has been found also to modulate a variety of functions of immune system cells in vitro and in vivo. IL-2 is one of several lymphocyteproduced messenger-regulatory molecules which mediate immunocyte interactions and functions.
IL-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for example, U.S. Pat. No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi, T., et al., Nature (1983) 302:305-310 and Devos, R., Nucleic Acids Research (1983) 11:4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms.
Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNA's, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon (.beta.-HIFN) is generally produced by superinducing human fibroblast cultures with poly-IC (polyriboinosinic acid and polyribocytidylic acid) and isolating and purifying the .beta.-HIFN thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon like properties may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-RNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the .beta.-HIFN therefrom. Nagola, S. et al., Nature, 284:316 (1980); Goeddel, D. V. et al., Nature, 287:411 (1980); Yelverton, E. et al., Nuc. Acid Res., 9:731 (1981); Streuli, M. et al., Proc. Nat'l. Acad. Sci. (U.S.), 78:2848 (1981); European Pat. Application Numbers 28033, published May 6, 1981; 321134, published July 15, 1981; 34307 published Aug. 26, 1981; and Belgian Patent 837397, issued June 1, 1981 describe various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFN's thus appear to have potential therapeutic use as anti-viral and anti-tumor agents and the production of IFN's by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost for clinical testing.
Protein samples for use in clinical studies, however, rust be of relatively high purity and substantially uncontaminated with toxic host cell constituents, cell debris and other extraneous chemicals introduced during the extraction and purification steps. There are several methods currently available for the preparation, recovery and purification of bacterially produced proteins.
U.S. Pat. No. 4,315,852 to Leibowitz et al. describes and claims a method for the acid extraction of leukocyte interferon from bacterial cells and neutralization of the extractant to obtain the interferon.
Derynck et al., Nature, 287:193 (1980) teach lysing transformed E. coli cells using a solution containing 5M urea, 1% sodium dodecyl sulfate (SDS), and 1% 2-mercaptoethanol. The lysate, which was purified by chromatography, exhibited interferon activity.
Scandella and Kornberg, Biochemistry, 10:4447 (1971) describe the preparation of a phospholipase from E. coli by solubilizing the cell membranes with SDS and precipitating the solubilized protein with 1-butanol.
U.S. Pat. No. 4,343,735, to Menge et al. teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U.S. Pat. No. 4,343,736 to Uemura et al. discloses a method for recovering interferon by absorption on water-insolubilized heparin and then eluting the interferon with an aqueous solution of an inorganic salt and chondroitin sulfate.
U.S. Pat. No. 4,289,689 to Friesen et al. discloses how to recover and purify human native .beta.-interferon by use of affinity chromatography and high pressure liquid chromatography.
U.S. Pat. No. 4,460,574 to Yabrov discloses a pharmaceutical composition comprising native human and.alpha.- and .beta.-interferons used for rectal or urogenital treatment of human interferon-sensitive diseases.
U.S. Pat. No. 4,364,863 to Leibowitz et al. describes a method of extracting fibroblast interferon from bacteria using a low pH followed by a high pH extraction procedure.
PCT WO 80/02229 to Benzon discloses purification of alpha (leukocyte) interferon, which is not a lipophilic protein.
EP 42,246 discloses that recombinant interferons may be dissolved in any pharmaceutically acceptable non-toxic carrier appropriate for the desired form of administration without further details.
U.S. Pat. No. 4,450,103 discloses solubilizing the protein in an aqueous medium with an appropriate solubilizing agent, extracting the protein from the aqueous medium with 2-butanol or 2-methyl-2-butanol, and precipitating the protein from the alcohol phase.
Cancer Treatment Reports, 62, 1900-1906 (1978) and EP 89,245 disclose that native beta-interferon may be formulated directly with human serum albumin in a pharmaceutically compatible aqueous-based medium at a pH of 7.2-7.8.
Alpha-interferons and native beta-interferon are not lipophilic proteins. Therefore, they can be stabilized and solubilized by adding a stabilizer such as human serum albumin directly to the formulation at physiological pH. In contrast, lipophilic proteins such as recombinant beta-interferon and interleukin-2 are not solubilized by addition of human serum albumin at pH 6.8-7.8.
A major problem with the existing methods of purification and recovery of lipophilic proteins is that the protein is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes, and further, that the resulting protein preparations, especially those that are produced by recombinant DNA techniques, have residual toxic amounts of chemicals, such as SDS and other surfactants or precipitants used in the extraction and purification steps. Thus, these preparations are not acceptable for clinical studies designed to determine the extent of the therapeutic use and applications of these proteins. It would be desirable, therefore, to have available a process for the recovery of a lipophilic protein in sufficiently large quantities and without toxic levels of SDS for clinical and therapeutic applications.
Applicants have heard of a disclosure in early 1985 of low pH formulation of proteins, but have no evidence thereof.
Accordingly, it is an object of the present invention to provide a pharmaceutically acceptable sample of a lipophilic protein such as recombinant .beta.-interferon or interleukin-2 which is of relatively high purity.
Another object of the present invention is to provide pharmaceutically acceptable samples of lipophilic proteins such as recombinant .beta.-interferon or interleukin-2 in sufficiently large quantities for clinical and therapeutic applications.
Yet another object of the instant invention is to provide lipophilic proteins such as recombinant .beta.-interferon or interleukin-2 preparations which are substantially free of SDS without loss of their biological activity, or at levels that are therapeutically acceptable.
A further object of this invention is to provide recombinant .beta.-interferon and interleukin-2 samples wherein the level of SDS is less than about 10 p.p.m.